B22-Multimode InfraRed imaging And Microspectroscopy
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Hany M.
Elsheikha
,
Alaa T.
Al-Sandaqchi
,
Mohammad S. R.
Harun
,
Francesca
Winterton
,
Ali
Altharawi
,
Nashwa A.
Elsaied
,
Carl W.
Stevenson
,
William
Macnaughtan
,
John G. M.
Mina
,
Paul W.
Denny
,
Gianfelice
Cinque
,
Ka Lung Andrew
Chan
Diamond Proposal Number(s):
[20222]
Open Access
Abstract: oxoplasma gondii (T. gondii) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by T. gondii (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of T. gondii-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to T. gondii infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during T. gondii infection.
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Feb 2022
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[18680, 20906, 22347, 23081]
Abstract: Synchrotron FTIR microspectroscopy coupled with mass spectrometric analysis of desorbed products has been used to investigate the initial stages of the methanol to olefins (MTO) reaction in single crystals of SAPO-34. Surface methoxy groups (SMS) are key to initial dimethylether (DME) and subsequent carbon–carbon bond formation. Deprotonation of SMS is the critical first step in direct olefin formation at low temperatures and DME is not involved in the carbon–carbon forming step. Experiments with CD3OH confirm the deprotonation step and show an inverse kinetic isotope effect consistent with irreversible deprotonation. The subsequent formation of alkoxide species, which are the precursors of the olefinic hydrocarbon pool present in working MTO catalysts, is initiated via insertion of surface carbene-like species into adjacent SMS. The observed induction period for this process is determined by the limited mobility of SMS and/or carbene species. Olefins formed from cracking of the alkoxide species then transmit carbon–carbon bond formation through the SAPO-34 by rapid diffusion and reaction with further SMS. Acetyl species seen with methanol at higher temperatures support the insertion of CO into SMS suggested in the literature, but these species do not play a role in direct olefin formation.
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Feb 2022
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[18811]
Open Access
Abstract: We used infrared (IR) microscopy to monitor in real-time the metabolic turnover of individual mammalian cells in morphologically different states. By relying on the intrinsic absorption of mid-IR light by molecular components, we could discriminate the metabolism of adherent cells as compared to suspended cells. We identified major biochemical differences between the two cellular states, whereby only adherent cells appeared to rely heavily on glycolytic turnover and lactic fermentation. We also report spectroscopic variations that appear as spectral oscillations in the IR domain, observed only when using synchrotron infrared radiation. We propose that this effect could be used as a reporter of the cellular conditions. Our results are instrumental in establishing IR microscopy as a label-free method for real-time metabolic studies of individual cells in different morphological states, and in more complex cellular ensembles.
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Oct 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[21061]
Abstract: Detecting metabolic changes inside a biological cell as a result of drug treatment is fundamental in pharmaceutical development. Currently, most techniques require the addition of dye (staining) to reveal some chemical changes, which may alter the natural process and potentially produce misleading results. Infrared (IR) microspectroscopy can probe chemical changes in biological matter
without the use of dye or chemical label, but the images produced optically so far are too coarse to see clearly inside a single cell.
Macrophages are a type of white blood cell that defend against infection. They produce many tiny fatty droplets when exposed to drugs, but the exact reason for this response was not clear. Knowing the chemical composition of these droplets will help scientists better understand the process and could help develop better medicines.
A team of researchers investigated a new Synchrotron IR spectroscopic imaging method, developed on Diamond Light Source’s Multimode InfraRed Imaging And Microspectroscopy (MIRIAM) beamline (B22), to probe the molecular changes inside macrophages. The results showed that IR nanospectroscopy can measure a drug’s effect inside a single mammalian cell by clearly identifying the chemical changes within the tiny fatty droplets before and after the application of the drug. Researchers will be able to use this powerful new molecular imaging tool to understand the responses of macrophages exposed to different drugs. This will help to identify new drug candidates that are more suitable for further development versus those that are for example ineffective or toxic, improving the chance of success in delivering better medicines in the near future.
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Jul 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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Philip A.
Ash
,
Sophie E. T.
Kendall-Price
,
Rhiannon M.
Evans
,
Stephen
Carr
,
Amelia
Brasnett
,
Simone
Morra
,
Ricardo
Hidalgo
,
Adam J.
Healy
,
Gianfelice
Cinque
,
Mark D.
Frogley
,
Fraser A
Armstrong
,
Kylie A.
Vincent
Diamond Proposal Number(s):
[17753, 19269, 21651]
Open Access
Abstract: Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) from Escherichia coli makes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectra in situ from a small area of crystal. The output reports on active site speciation via the vibrational stretching band positions of the endogenous CO and CN- ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle.
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Jun 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[21620]
Abstract: The present experimental work applied coherent synchrotron-radiation THz spectroscopy and inelastic neutron scattering to address two processes directly associated with the mode of action of metal-based anticancer agents, that can severely undermine chemotherapeutic treatment: drug binding to human serum albumin, occurring during intravenous drug transport, and intracellular coordination to thiol-containing biomolecules (such as metallothioneins), associated with acquired drug resistance. Cisplatin and two dinuclear Pt- and Pd-polyamine agents developed by this research group, which have yielded promising results towards some types of human cancers, were investigated. Complementary SR-THz and INS data revealed protein metallation, through S- and N-donor ligands from cysteine, methionine and histidine residues. A clear impact of the Pt- and Pd-agents was evidenced, drug-binding to albumin and metallothionein having been responsible for significant changes in the overall protein conformation, as well as for an increased flexibility and possible aggregation.
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Jun 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[11425]
Abstract: In this work, we address the kinetics of dehydrogenation occurring at high temperatures (HT) in riebeckite, a sodic amphibole with the ideal composition Na2Fe3+2 Fe2+3Si8O22(OH)2. We did isothermal experiments on both powders and single-crystals up to 560 °C and monitored the O-H stretching signal by Fourier Transform Infrared (FTIR) spectroscopy. Single-crystals show an initial increase in IR absorption intensity due to increasing vibrational amplitudes of the O-H bond stretching, not observed for powders. The OH-intensities vs. time were fitted using the formalism for first-order reactions. The calculated activation energies for H+ diffusion in riebeckite are 159 ± 15 kJ/mol for powders and 216 ± 20 kJ/mol for single crystals, respectively. The exponential factor m in the Avrami-Erofeev equation obtained for crystals ranges between 1.02 and 1.31, suggesting that, unlike powders, the dehydration process in crystals is not a purely first-order reaction. This implies that a second energy barrier must be considered, i.e., diffusion of H+ through the crystal. FTIR imaging showed that H+ diffusion occurs mainly perpendicular to the silicate double-chain. Our results confirm that the release of H+ from riebeckite occurs after the irreversible Fe2+-to-Fe3+ exchange, thus at temperatures > 550 °C. To be effective, the process needs the presence of external oxygen that, by interacting with H+ at the crystal surface, triggers the release of H2O molecules. This implies that oxidizing conditions are required for the amphibole to be an efficient water source at depth.
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May 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
I11-High Resolution Powder Diffraction
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Lydia
Briggs
,
Ruth
Newby
,
Xue
Han
,
Christopher
Morris
,
Mathew
Savage
,
Cristina
Perez
,
Timothy L.
Easun
,
Mark
Frogley
,
Gianfelice
Cinque
,
Claire A.
Murray
,
Chiu C.
Tang
,
Sihai
Yang
,
Junliang
Sun
,
Martin
Schroeder
Diamond Proposal Number(s):
[22137, 22138]
Open Access
Abstract: We report the adsorption of C2H2, CO2 and SO2 in a new, ultra-stable Cr(III)-based MOF, MFM-300(Cr), {[Cr2(OH)2(L)], H4L = biphenyl-3,3',5,5'-tetracarboxylic acid}. MFM-300(Cr) shows uptakes of 7.37, 7.73 and 8.59 mmol g-1 for CO2, C2H2 and SO2, respectively, at 273 K, 1.0 bar, and shows a higher selectivity for SO2/CO2 compared with the Al(III) analogue MFM-300(Al) (selectivity of 79 vs. 45). In order to monitor the effects of changing metal centre on gas uptake and to integrate the properties of the homometallic analogues, the mixed metal MFM-300(Al0.67Cr0.33), [Al1.34Cr0.66(OH)2L] has been synthesised. In situ synchrotron micro-FTIR spectroscopy has identified distinct CO2 binding environments on Al-O(H)-Al, Cr-O(H)-Cr and Al-O(H)-Cr bridges in MFM-300(Al0.67Cr0.33), and we have determined the binding domains for these gases by in situ synchrotron X-ray diffraction in both MFM-300(Cr) and MFM-300(Al0.67Cr0.33). The capability of these materials for gas separation has been confirmed by dynamic breakthrough experiments. The incorporation of Al(III) and Cr(III) within the same framework allows tuning of the host-guest and guest-guest interactions within these functional porous materials.
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Feb 2021
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B22-Multimode InfraRed imaging And Microspectroscopy
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James M.
Cameron
,
Justin J. A.
Conn
,
Christopher
Rinaldi
,
Alexandra
Sala
,
Paul M.
Brennan
,
Michael D.
Jenkinson
,
Helen
Caldwell
,
Gianfelice
Cinque
,
Khaja
Syed
,
Holly J.
Butler
,
Mark G.
Hegarty
,
David S.
Palmer
,
Matthew J.
Baker
Diamond Proposal Number(s):
[23417]
Open Access
Abstract: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are found in a high proportion of diffuse gliomas. The presence of the IDH1 mutation is a valuable diagnostic, prognostic and predictive biomarker for the management of patients with glial tumours. Techniques involving vibrational spectroscopy, e.g., Fourier transform infrared (FTIR) spectroscopy, have previously demonstrated analytical capabilities for cancer detection, and have the potential to contribute to diagnostics. The implementation of FTIR microspectroscopy during surgical biopsy could present a fast, label-free method for molecular genetic classification. For example, the rapid determination of IDH1 status in a patient with a glioma diagnosis could inform intra-operative decision-making between alternative surgical strategies. In this study, we utilized synchrotron-based FTIR microanalysis to probe tissue microarray sections from 79 glioma patients, and distinguished the positive class (IDH1-mutated) from the IDH1-wildtype glioma, with a sensitivity and specificity of 82.4% and 83.4%, respectively. We also examined the ability of attenuated total reflection (ATR)-FTIR spectroscopy in detecting the biomolecular events and global epigenetic and metabolic changes associated with mutations in the IDH1 enzyme, in blood serum samples collected from an additional 72 brain tumour patients. Centrifugal filtration enhanced the diagnostic ability of the classification models, with balanced accuracies up to ~69%. Identification of the molecular status from blood serum prior to biopsy could further direct some patients to alternative treatment strategies.
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Dec 2020
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B22-Multimode InfraRed imaging And Microspectroscopy
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Diamond Proposal Number(s):
[15393]
Open Access
Abstract: Glycosaminoglycans (GAGs)/proteoglycans (PGs) play a pivotal role in the metastasis of inflammatory breast cancer (IBC). They represent biomarkers and targets in diagnosis and treatment of different cancers including breast cancer. Thus, GAGs/PGs could represent potential prognostic/diagnostic biomarkers for IBC. In the present study, non-IBC MDA-MB-231, MCF7, SKBR3 cells and IBC SUM149 cells, as well as their GAG secretome were analyzed. The latter was measured in toto as dried drops with high-throughput (HT) Fourier Transform InfraRed (FTIR) spectroscopy and imaging. FTIR imaging was also employed to investigate single whole breast cancer cells while synchrotron-FTIR microspectroscopy was used to specifically target their cytoplasms. Data were analyzed by hierarchical cluster analysis and principal components analysis. Results obtained from HT-FTIR analysis of GAG drops showed that the inter-group variability enabled us to delineate between cell types in the GAG absorption range 1350–800 cm−1. Similar results were obtained for FTIR imaging of GAG extracts and fixed single whole cells. Synchrotron-FTIR data from cytoplasms allowed discrimination between non-IBC and IBC. Thus, by using GAG specific region, not only different breast cancer cell lines could be differentiated, but also non-IBC from IBC cells. This could be a potential diagnostic spectral marker for IBC detection useful for patient management.
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Sep 2020
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